The invention relates to novel indole-derived compounds and their use in the preparation of a medicine of use in the treatment of diseases related to the splicing process.
Certain indole-derived compounds, such as ellipticine and aza-ellipticine derivatives, are already known as intercalating molecules which correct errors in genetic expression during the replication process. They have been more specifically described for the treatment of diseases such as cancer, leukemia and AIDS (FR 2 627 493, FR 2 645 861, FR 2 436 786).
The intracellular splicing process consists of eliminating pre-messenger RNA introns in order to produce mature RNA messengers which can be used by the cell's translation machinery (Sharp, P. A. (1994). Split genes and RNA splicing. Cell 77, 805-815). In the case of alternative splicing, the same precursor can be the source for messenger RNAs that code for functionally-distinct proteins (Black, D. L. Mechanisms of Alternative Pre-Messenger RNA Splicing. Annu. Rev. Biochem. 2003. 72, 291-336). The precise selection of the 5′ and 3′ splice sites is thus a mechanism which generates diversity and which can lead to the regulation of gene expression as a function of tissue type and over the course of an organism's development. The factors implicated in this selection include a family of proteins termed SR (serine/arginine-rich protein) characterized by the presence of one or two RRM (RNA-recognition motif) RNA-binding domains and a domain termed RS (arginine/serine) rich in arginine and serine residues (Manley, J. L. and Tacke, R. (1996). SR proteins and splicing control. Genes Dev. 10, 1569-1579). By binding to the pre-mRNA's short exonic and intronic sequences, termed ESE (exonic splicing enhancer) and ISE (intronic splicing enhancer), SR proteins are capable of dose-dependently activating suboptimal splicing sites and enabling the inclusion of exons (Graveley, B. R. Sorting out the complexity of SR protein functions. RNA. 2000. 6, 1197-1211). SR protein activity in alternative splicing is specific insofar as the inactivation of the corresponding gene is lethal (Wang, H. Y. et al., SC35 plays a role in T cell development and alternative splicing of CD45. Mol. Cell 2001. 7, 331-342).
Sequencing of the human genome and analysis of EST (expressed sequence tag) collections has revealed that 35 to 65% of genes are expressed in the form of alternative splicing variants (Ewing, B. and Green, P. Analysis of expressed sequence tags indicates 35,000 human genes. Nat. Genet. 2000. 25, 232-234). This mechanism is thus a favored target of modifications which can affect the factors implicated in the regulation of splicing and of mutations which affect the sequences necessary to this regulation. At present, it is estimated that approximately 50% of the point mutations responsible for genetic diseases result in splicing errors. These mutations can interfere with splicing by inactivating or creating splicing sites, but also by modifying or generating regulation elements known as “splicing enhancers” and “splicing silencer” in a specific gene (Cartegni, L. et al., Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat. Rev. Genet. 2002. 3, 285-298).
The strategies currently developed to correct these splicing errors are based on the use of various types of molecules.
The document by Tazi J et al. (DNA topoisomerase I: customs officer at the border between DNA and RNA worlds?, Journal of Molecular Medecine (Berlin, Germany) 1997 November-December, vol. 75, no. 11-12) describes that certain derivatives inhibit DNA topoisomerase I, a specific kinase which phosphorylates SR splicing factors.
The document by Poddevin B et al. (Dual topoisomerase I and II inhibition by intoplicine (RP-60475), a new antitumor agent in early clinical trials, Molecular Pharmacology, Baltimore, Md., US, vol. 44, no. 4) describes that intoplicine is a molecule that inhibits both topoisomerase I and topoisomerase II, demonstrating that this compound can be active against tumors.
The document by Pilch B et al. (Specific inhibition of serine- and arginine-rich splicing factors, phosphorylation, spliceosome assembly, and splicing by the antitumor drug NB-506, Cancer Research, 15 Sep. 2001, US, vol. 61, no. 18) describes that indolocarbazole medicines inhibit topoisomerase I and can as a result be considered anti-cancer medicines.
One strategy targeting the development of new molecules that make it possible to correct or eliminate splicing errors, for example, is based on the overexpression of proteins that interfere with this type of splicing (Nissim-Rafinia, M. et al., Cellular and viral splicing factors can modify the splicing pattern of CFTR transcripts carrying splicing mutations. Hum. Mol. Genet. 2000. 9, 1771-1778; Hofmann, Y. et al., Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc. Natl. Acad. Sci. U.S.A. 2000. 97, 9618-9623).
Another strategy is based on the use of antisense oligonucleotides (Sazani, P. et al., Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nat. Biotechnol. 2002. 20, 1228-1233; Sazani, P. and Kole, R. Modulation of alternative splicing by antisense oligonucleotides. Prog. Mol. Subcell. Biol. 2003. 31, 217-239) and of PNA (peptidic nucleic acid) (Cartegni, L. et al., Correction of disease-associated exon skipping by synthetic exon-specific activators. Nat. Struct. Biol. 2003. 10, 120-125) making it possible to inhibit or activate, respectively, a splicing event.
Still another strategy is based on the identification of compounds that influence the effectiveness of splicing of the pre-mRNA of interest (Andreassi, C. et al., Aclarubicin treatment restores SMN levels to cells derived from type I spinal muscular atrophy patients. Hum. Mol. Genet. 2001. 10, 2841-2849).
Finally, a strategy based on the use of trans-splicing to replace mutated exons has been described (Liu, X. et al., Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat. Biotechnol. 2002. 20, 47-52).
One of the disadvantages of the strategies developed and cited above to correct or eliminate splicing errors is their cost of production. Indeed, the cost of producing antisense oligonucleotides that must be modified to improve their stability, and also the cost of producing PNA molecules, is high.
Another disadvantage of the strategies developed and cited above is that they require the use of expression vectors, as for example for the strategy based on the use of trans-splicing.